The invention relates to a method and an agent for the determination of maleimide groups and in particular for the determination of reactive maleimide groups bonded to proteins, e.g., enzymes, antigens and antibodies.
Since the mid-1970's, many so-called heterocrosslinkers have been developed for the purpose of covalently bonding proteins together via different reactive groups, e.g., amino groups, carboxyl groups, hydroxyl groups and thiol groups. These heterocrosslinkers have become of particular interest in the area of immunoassay reagents. Heterocrosslinkers carrying maleimide groups may be specially singled out here because they have the property of reacting specifically with thiol groups. Thus, antibody fragments, e.g., Fab' fragments, containing free thiol groups can be selectively bonded to other proteins, e.g., enzymes.
Examples of such crosslinkers and heterocrosslinkers carrying maleimide groups are succinimidyl m-maleimidobenzoate (SMB), sulfosuccinimidyl m-maleimidobenzoate (sulfo-SMB), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB), bis-maleimidohexane (BMH), N-(4-diazophenyl)maleimide and N-(.beta.-diazophenylethyl)maleimide.
Thiol groups can be bonded to amino groups with the aid of these heterocrosslinkers. Furthermore, N-(4-diazophenyl)maleimide and N-(.beta.-diazophenylethyl)maleimide also make it possible to bond thiol groups to imidazole, phenol, indole and other groups.
In principle, these reactions take place according to the following scheme: EQU R--NH.sub.2 +succ--O--CO--X--mal.fwdarw.R--NH--CO--X--mal+succ--OH (1st step) (I) EQU R--NH--CO--X--mal+HS--R'.fwdarw.R--NH--CO--X--malH--S--R'(2nd step)(II)
wherein R--NH.sub.2 = a protein-carrying primary amino groups; mal =a maleimide group; succ = a succinimidyl group; R'--SH =a protein-carrying thiol group; and X=a spacer.